Identification of Alkaloid’s Profile in Ficus benjamina L. Extracts with Higher Antioxidant Power

Silvia Novelli, Canuti Lorena, Canini Antonella

Department of Biology, University of Rome "Tor Vergata", Rome, Italy.

DOI: 10.4236/ajps.2014.526421                

Abstract

Different classes of known alkaloid compounds were identified for the first time in leaves and barks of Ficus benjamina L. from Moraceae family. The identification of 28 alkaloids in F. benjamina leaves and 14 alkaloids in its barks was identified using chromatographic analysis as Gas Chromatography associated to Mass Spectrometry (GC-MS). This characterization analysis allows to verifying the presence of different alkaloids type-classes in both leaves and barks, such as isoquinoline class, indole class and quinolizidine class. The quantification results are deduced by analysis with spectrophotometric assay, using a calibration curve of boldine as standard alkaloid that reveals the presence of 26 ± 0.1 μg of alkaloids boldine equivalent in 1 gram of F. benjamina leaves and 3.8 ± 0.1 μg alkaloids boldine equivalent in 1 gram of its barks. A significant correlation was observed between the total alkaloid content of crude extracts and 2,2-diphenyl-1-picrylhydrazyl assay (DPPH), suggesting that the level of antioxidant activity in this species is strongly correlated to the alkaloid content. These findings should provide useful information for future on the pharmaceutical properties of these alkaloids from F. benjamina.

Keywords

Ficus benjamina, Alkaloids, Columbamine, Antioxidant Power

Introduction

Health assessment is defined as an evaluation of the health status of an individual by performing symptom assessment, physical examination and history inquiry (Day, 2010). When nurses interact with patients and provide care, the appropriate assessment skills with obtaining a complete health history are critical to identify physical and psychological problems and concerns experienced by patients. Nurses should consider pa- tients’ educational and cultural background and their language proficiency, and pay close attention to the patient’s disabilities or impairments in hearing, vision, cognitive and physical limi- tations during health assessment (Pan, 2012). Although assess- ment is an essential competency of nurses, little has been ac- tually done by nurses in clinical settings. Secrest, Norwood, & duMont’s (2005) survey showed that although 92.5% of physi- cal assessment skills were taught in baccalaureate nursing pro- gram, only 29% of nurses in clinical practice actually per- formed those skills. Liu, Chen and Yang (2008) indicated that the assessment skill deficiencies of new nurses included lack of communication skills, incorrect physical examination technique, and lack of comprehensive assessment and analysis of patient condition. A gap may exist between what is taught in classroom and what is actually performed in nursing practice. In Macao, students usually prefer to learn practical knowledge in an organized environment. The static manikins commonly have

been delivered under the direction or demonstration of teachers. Students had less opportunity to learn or practice the skills in an interactive environment. Preparation of students for a complex health care environment requires that students are educated not only in skills but also in communication and collaborative care. An ongoing concern with nursing education is how to improve students’ assessment skills using appropriate strategies as well as knowledge application (Mei, He, Xie, Yang, & Duan, 2012). Students should learn how to solve problems and practice over and over in each trial while accepting and learning from their mistakes until they become skilled. In this case, more active teaching strategies, such as role play, case study, or standar- dized patient teaching method, were used in course learning of health assessment and showed that the experimental group got the higher scores in both theory and skill exams than the control group who served as a traditional method (Chen, Cheng, Zhou, Feng, & Dong, 2012; Pan, 2012; Yin et al., 2012).

Although learning by doing is a long established means for facilitating knowledge acquisition, it isn’t practical to engage in skill training with real patients due to the increased protection of patient rights and safety. The complexity of the current health care systems makes it difficult to provide nursing stu- dents with sufficient clinical experiences to ensure their com- petency. Educators are challenged to find adequate clinical experiences for their students. Simulation provides the oppor- tunity for the learner to practice and learn in an environment as

close to reality as possible and allows students to construct knowledge and develop psychomotor skills in a safe environ- ment (Sinclaire & Fergusion, 2009). Students interact with a human patient simulator and discover critical assessment in- formation in the same manner they would with real patients. They had an opportunity to rehearse skills in a simulated envi- ronment without fear of failure or compromising patient safety (Bambini, Washburn, & Perkins, 2009).

As an experience learning, simulation using a human patient simulator (the SimMan) which imitates patient condition with physiological functions can be used for training health assess- ment skills as well as communication skills (Haidar, 2009). However, little current study discussed how to improve as- sessment skills and knowledge application of medical-surgical nursing using simulation in nursing students. The observational measure was used in this study to discuss about the research question as a difference in health assessment scores followed by each simulation session.

Research Objective

The objective of this study was to determine how students’ assessment scores changed at a simulated setting followed by each simulation session.

Method

A quasi experimental study with one group repeated-meas- ures (from Session 1 to Session 5) design was conducted at one nursing school in Macao. The scores measured in each simula- tion session were compared within each tutorial group.

Samples

Purposive sampling was used to recruit Year 2 and Year 3 baccalaureate nursing students who passed course learning in health assessment and medical-surgical nursing. Totally there were 115 students (54 in Year 2, 61 in Year 3). 90 of them (52 in Year 2, 38 in Year 3) voluntarily participated in this study. Finally 85 students (49 in Year 2, 36 in Year 3) completed all simulation sessions. Their average age was 20.24 (S.D. 1.46) years old. Second-year students had 20 weeks of clinical expe- rience while third-year students had 30 weeks of experience.

Teaching Protocol

The 36-hour simulation training was conducted as extracur- ricular activities for promoting students’ health assessment skills. The scenarios were designed by two qualified tutors using a human patient simulator which is a computer-controlled full-body manikin with a realistic upper airway, chest move- ment, variable cardiac and breath sounds and a palpable pulse. In each grade, 5 or 6 students worked in a group. Students di- rectly interacted with a contextual patient scenario and per- formed health assessment adhering to principles of safety. Role play was used in “nurse-patient” communication. One student served as a patient or a family member or a friend who pro- vided personal information while the other students would be the nurses who provided patient care. The tutors acted as facili- tators to promote students’ learning. Each group simulation was video-recorded for evaluating students’ performance in health assessment. Table 1 showed the teaching protocol in each ses- sion.

Instrument

Based on the Day’s (2010) health assessment guidelines, the health assessment evaluation rubric (NAER) (an observational measurement) was developed by two qualified tutors (see Ta- ble 2). The face validity was approved by three experts in health assessment and medical-surgical nursing. It was used to evaluate students’ performance in health assessment in terms of introduction and patient identification (2 items), symptom as- sessment (2 items), physical examination (6 items), patient education (1 item), history inquire (1 item) and communication (2 items). It is ranked as three levels (2 excellent, 1 satisfactory, 0 needs practices). The possible score ranges from 0 to 28. A higher score indicates better performance in health assessment (see Table 2).

In this study, the NAER was completed by two qualified tu- tors for assessing the performance of each group. The inter- rater reliabilities of the NAER were 0.818 (Session 1, P < 0.01), 0.814 (Session 2, P < 0.01), 0.812 (Session 3, P < 0.01), 0.801 (Session 4, P < 0.01) and 0.928 (Session 5, P < 0.01).

In addition, one open-end question (what do you think of si- mulation in promoting your assessment skills?) was used to describe students’ perceptions about the impact of simulation experience on the development of health assessment.

Date Collection and Analysis

Health assessment skills were evaluated at the end of each simulation session prior to debriefing. Two tutors completed the scoring of the NAER for each group and provided the ra- tionale for each score assigned. Differences in interpretation were discussed and negotiated until similar rational for scores given could be verbalized. The average scores of two tutors were calculated. The repeated measures analysis of variance (RM-ANOVA) was used to assess differences of health as- sessment within groups as well as across time for simulation. Independent samples t-test was carried out to compare the mean scores of the NAER between Year 2 and Year 3 students. Addi- tionally, the open-ended question was completed at the end of the last simulation session. All responses were summarized by their meanings with analysis of frequency.

Ethical Considerations

The research process was discussed by the workgroup for academic affaires and approved by the board of management of the institute. Participants were provided with a complete expla- nation about the objective and process of the study. The written consent from each participant was obtained. All participants were entirely voluntary and had the freedom to withdraw from the study at any time. Confidentiality and autonomy were as- sured. Only aggregate data would be reported.

Results

The overall score of assessment skills increased from the first session to the last session, especially in communication and symptom assessment (see Table 3). Second-year students achieved higher overall scores of assessment skills in Session 2, 4 and 5 than third-year students significantly. They presented better physical examination in each session, and better communication and patient education in Sessions 3, 4 and 5 (see Table 4).

Table 1.The teaching protocol in each simulation session.

Regarding students’ written comments, students indicated that simulation enhanced their auscultation skills (70.6%) and their abilities to prioritize the problems (78.8%), to assess the client systematically (76.5%), to manage contingencies and emergencies (74.1%), to collaborate with others effectively (50.6%). However, 71.8% students considered it was difficult to emulate the lived experience using the SimMan because of the slow response to inquiries and no facial expression.

Discussion

The findings indicated that simulation using a human patient simulator facilitated students’ health assessment skills, espe- cially in communication and symptom assessment. It is possi- bly caused by scenario design and learning activities in simula- tion. The scenarios used in this study were designed to reveal the ability of students to make sense of data, not only in how to

Table 2.Nursing assessment evaluation rubric.

Table 3. Changes of mean scores in nursing assessment (N = 85).

Table 4. Comparison of scores of health assessment skills between Year 2 and Year 3 students in each session.

assess the patients and set priorities but also in how to provide patient education on complex topics. Students used analytic thinking and clinical reasoning processes to interpret the mean- ings of obtained data, and chose the appropriate response to patient condition meanwhile they also need communicate with the patient in understandable way. The interaction with the realistic scenarios enabled students to “understand” patient’s feelings and realize the severity and urgency of patient condi- tions, and “read” the patient’s responses to the intervention. This experience can help students to bridge the theory practice gap by transferring cognitive learning into practical experience. Health assessment skills were increased by assessment of the relevant data, a logical interpretation and reasoning and accu- rate judgments.

The reflections offered a unique way for students to critically analyze their own performance. Students engaged in introspec- tive learning to self-correct. The reflections focused on students’ primary misconceptions, anything they missed in report or oth- er information they needed from report or the patient to act more effectively, and what they should do differently the next time while emphasizing what was correct, appropriate and safe. It allows the student to clarify their thinking and link the simu- lation to real situation while reinforcing specific knowledge, and to discuss how to intervene professionally in complex clin- ical situations (Gaberson & Oermann, 2010). In this case, stu- dents learned from previous experience and paid close attention to patients’ concerns. They assessed the relevant and important data and explained them to the patient using understandable wording as managing the contingencies and emergencies. They presented better communication skills and patient education across the time of simulation.

Previous studies reported the consistent findings. Kaddoura (2010) reported that simulation prepared new nursing graduates well to care confidently for critically ill patients, and helped them learn to make sound clinical decisions to improve patient outcomes. Zheng et al. (2010) found that students’ performance was significantly improved in application of theoretical know- ledge, health education and humanistic care after one-semester of simulation. More than 95% of students agreed that feedback sessions confirmed management of patients’ problems, helped to develop rationale for actions (Wotton, Davis, Button, & Kelton, 2010).

However, students indicated that the SimMan is not realistic enough. The SimMan had its own inherent limitation. It may do not duplicate the experience of working with a live patient. By responding to a situation during the scenario, the “patient” pro- vided instant feedback; through which students saw the out- comes of their interventions. It was suggested that forthright feedback from the facilitator was needed to enhance the realism of the scenario with physical props and psychosocial interac- tions (Birkhoff & Donner, 2010).

The interesting finding in this study was that second-year students achieved higher overall scores of health assessment in some sessions than third-year students. They presented better physical examination in each simulation session, and better communication and patient education in some sessions. It may be caused by the different learning effort of students. The tutors’ comments showed that second-year students valued the newly learned knowledge and applied it in the simulated scenarios. They did good preparation for learning and engaged in group learning, deep discussion and reflection. They try their best to make the physical examination comprehensive while concern- ing the patient’s response and providing the appropriate man- agement. However, third-year students did not have a deep memory and understanding of some knowledge that they learned in their previous two years, and did not do a full know- ledge review and a good skill preparation for the simulated learning. Their assessment was not comprehensive while their explanations to abnormal sign and symptoms were incorrect or ambiguous. Sometime they could not recognize some severe arrhythmias. Thereby, they got the lower scores in physical examination, patient education and communication compared with the second-year students. For ensuring the quality of learn- ing, students should have good preparation for knowledge and skills, be self-motivated and keep responsible for their own learning. Tutors should promote students' intrinsic motivation for learning and develop their potential efforts in learning dur- ing simulation.

Limitations

The generalization of the findings was limited because a small purposive sample was recruited from one research setting. The new developed health assessment evaluation rubric was only used in medical-surgical care; the generalizability of fur- ther studies needs to be considered in other area of nursing care, such as long-term care or community care. As a confounding variable, the mixed role play of students (patients or family members and nurse) may affect the effective “nurse-patient” communication and thus influence the accuracy and scores of health assessment.

Recommendations

The performance indicators of the health assessment evalua- tion rubric require more research to address content and con- struct validity in different nursing contexts in order to more accurately reflect the current understanding of each aspect of health assessment. As transfer of skill from the simulated envi- ronment to the clinical setting is essential, follow-up studies need to be concerned with the impact of using simulation on students’ performance in clinical placement.

Conclusion

Simulation using a human patient simulator offered a realis- tic learning environment for students to develop their health assessment skills. Most of students appreciated that simulation facilitated their knowledge application, assessment and com- munication skills and group collaboration, but using manne- quins did not replace working with live patients. Forthright feedback from the facilitator was needed to enhance the realism of the scenario. Tutors should promote students’ intrinsic mo- tivation for learning and develop their potential and efforts in learning. The questionnaire needed to be carried out to investi- gate students’ perceptions about the impact of simulation expe- rience on the development of health assessment.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Acharya, D. and Shrivastava, A. (2008) Indigenous Herbal Medicine: Tribal Formulations and Traditional Herbal Practices. 1st Edition, Avishkar Publishers, Jaipur.
[2]	Aniszewski, T. (2007) Alkaloids—Secrets of Life Alkaloids Chemistry, Biological Significance Application and Ecological Role. 1st Edition, Elsevier, Amsterdam.
[3]	Podsedek, A. (2007) Natural Antioxidants and Antioxidant Capacity of Brassica Vegetables: A Review. Food Science and Technology, 40, 1-11.
[4]	Maestri, D.M., Nepote, V., Lamarque, A.L. and Zygadlo, J.A. (2006) Natural Products as Antioxidants. In: Trivandrum, I.F., Ed., Phytochemistry: Advances in Research, Research Signpost, Córdoba, 105-135.
[5]	Dang, S., Yan, H., Yamamoto, S., Wang, X. and Zeng, L. (2004) Poor Nutritional Status of Younger Tibetan Children Living at High Altitudes. European Journal of Clinical Nutrition, 58, 938-946. http://dx.doi.org/10.1038/sj.ejcn.1601915
[6]	Chaurasia, O.P., Ballabh, B. and Singh, B. (2003) Himalayan Rose Root, Rhodiola spp. Potential Health Drink and Drug Plant. Natural Product Radiance, 2, 71-92.
[7]	Ali, R., Jaimini, A., Nishad, D.K., Mittal, G., Chaurasia, O.P., Kumar, R., Bhatnagar, A. and Singh, S.B. (2012) Acute and Sub Acute Toxicity and Efficacy Studies of Hippophaerhamnoides Based Herbal Antioxidant Supplement. Indian Journal of Pharmacology, 44, 504-508. http://dx.doi.org/10.4103/0253-7613.99329
[8]	Hegedus, A., Engel, R., Abrankó, L., Balogh, E., Blázovics, A., Hermán, R., Halász, J., Ercisli, S., Pedryc, A. and Stefanovits-Bányai, é. (2010) Antioxidant and Antiradical Capacities in Apricot (Prunus armeniaca L.) Fruits: Variations from Genotypes, Years, and Analytical Methods. Journal of Food Science, 75, 722-730. http://dx.doi.org/10.1111/j.1750-3841.2010.01826.x
[9]	Yadav, A., Stobdan, T., Dwivedi, S.K., Korekar, G., Targais, K. and Srivastava, R.B. (2011) Apricot (Prunus armeniaca L.) for Livelihood Security in Cold Deserts. In: Srivastava, R.B. and Selvamurthy, W., Eds., Innovations in Agro Animal Technologies, Satish Serial Publishing House, Delhi, 87-97.
[10]	Chaurasia, O.P., Hota, S.K., Kumar, R., Tayade, A.B. and Srivastava, R.B. (2011) Trans-Himalayan Medicinal Plants and Herbal Nutraceutical Products. In: Srivastava, R.B. and Selvamurthy, W., Eds., Innovations in Agro Animal Technologies, Satish Serial Publishing House, Delhi, 113-129.
[11]	Kala, C.P. (2006) Medicinal Plants of the High Altitude Cold Desert in India: Diversity, Distribution and Traditional Uses. International Journal of Biodiversity Science & Management, 2, 43-56. http://dx.doi.org/10.1080/17451590609618098
[12]	Chaurasia, O.P., Ahmed, Z. and Ballabh, B. (2007) Ethnobotany and Plants of Trans-Himalaya. Satish Serial Publishing House, Delhi.
[13]	Liyana-Pathiranan, C.M. and Shahidi, F. (2005) Antioxidant Activity of Commercial Soft and Hard Wheat (Triticum aestivum L.) as Affected by Gastric pH Conditions. Journal of Agricultural and Food Chemistry, 53, 2433-2440. http://dx.doi.org/10.1021/jf049320i
[14]	Lansky, E.P., Paavilainen, H.M., Pawlus, A.D. and Newman, R.A. (2008) Ficus spp. (fig): Ethnobotany and Potential as Anticancer and Anti-Inflammatory Agents. Journal of Ethnopharmacology, 119, 195-213. http://dx.doi.org/10.1016/j.jep.2008.06.025
[15]	Baumgartner, B., Erdelmeier, C.A.J., Wright, A.D., Rali, T. and Sticher, O. (1990) An Antimicrobial Alkaloid from Ficusseptica. Phytochemistry, 29, 3227-3330. http://dx.doi.org/10.1016/0031-9422(90)80209-Y
[16]	Mohammad, H., Sutradhar, M.A. and Ranjrr, K. (1991) Chemical Constituents of Ficus glomerata Roxb. Journal of the Bangladesh Chemical Society, 4, 247-250.
[17]	Khan, I.A., Rali, T. and Sticher, O. (1993) Alkaloids from Ficus pachyrhachis. Planta Medica, 59, 286. http://dx.doi.org/10.1055/s-2006-959677
[18]	Ilyas, M. and Ilyas, N. (1990) Flavonoids from the Leaves of Ficus capensis. Ghana Journal of Chemistry, 1, 176-178.
[19]	Frith, H.J., Rome, F.H.J. and Wolfe, T.O. (1976) Food of Fruit-Pigeons in New Guinea. Emu, 76, 49-58. http://dx.doi.org/10.1071/MU9760049
[20]	Almahyl, H.A., Rahmani, M., Sukarp, M.A. and Ali, A.M. (2003) Investigation on the Chemical Constituents of the Leaves of Ficus elastica Roxb. and Their Antimicrobial Activity. Pertanika Journal of Science & Technology, 11, 57-63.
[21]	Parajuli, S.P. (2000) Ethnobotanical Studies of at Khandbarimuncipilty of Sankhuwasabha. BankoJanakari, 10, 29-34.
[22]	Sirisha, N., Sreenivasulu, M., Sangeeta, K. and Chetty, C.M. (2010) Antioxidant Properties of Ficus Species: A Review. International Journal of PharmTech Research, 4, 2174-2182.
[23]	Kanaujia, V.K., Rirchhaiya, H.K., Kailasiya, S.D., Verma, M., Yadav, R.D. and Shivhare, D. (2011) Evaluation of Hepatoprotective Activity on the Leaves of Ficus benjamina Linn. Journal of Natural Product & Plant Resources, 1, 59-69.
[24]	Dai, J., Shen, D., Yoshida, W.Y., Parrish, S.M. and Williams, P.G. (2012) Isoflavonoids from Ficus benjamina and Their Inhibitory Activity on BACE1. Planta Medica, 78, 1357-1362. http://dx.doi.org/10.1055/s-0032-1315001
[25]	Angelo, G., Lorena, C., Marta, G. and Antonella, C. (2014) Biochemical Composition and Antioxidant Properties of Lavandula angustifolia Miller Essential Oil Are Shielded by Propolis against UV Radiations. Photochemistry and Photobiology, 90, 702-708. http://dx.doi.org/10.1111/php.12229
[26]	Shamsa, F., Monsef, H., Ghamooshi, R. and Verdian-rizi, M. (2008) Spectrophotometric Determination of Total Alkaloids in Some Iranian Medicinal Plant. Thai Journal of Pharmaceutical Sciences, 32, 17-20.
[27]	Esmaeili, A.H., Moghaddam, A.H. and Chaichi, M.J. (2013) Identification, Determination, and Study of Antioxidative Activities of Hesperetin and Gallic Acid in Hydro-Alcoholic Extract from Flowers of Eriobotrya japonica L. Avicenna Journal of Phytomedicine, 4, 260-266.
[28]	Wong, S.K., Lim, Y.Y., Abdullah, N.R. and Nordin, F.J. (2011) Antiproliferative and Phytochemical Analyses of Leaf Extracts of Ten Apocynaceae Species. Pharmacognosy Research, 3, 100-106. http://dx.doi.org/10.4103/0974-8490.81957
[29]	Piana, M., Boligon, A.A., Brum, T.F., Zadra, M., Belke, B.V., Froeder, A.L., Frohlich, J.K., Nunes, L.T., Pappis, L., Boligon, A.A. and Athayde, M.L. (2014) Phytochemical Analysis and Antioxidant Capacity of Tabernaemontana catharinensis A. D.C. Fruits and Branches. Anais da Academia Brasileira de Ciências, Epub Ahead of Print.
[30]	Chow, Y. and Sato, F. (2013) Screening of Isoquinoline Alkaloids for Potent Lipid Metabolism Modulation with Caenorhabditis elagans. Bioscience, Biotechnology, and Biochemistry, 77, 2405-2412. http://dx.doi.org/10.1271/bbb.130523
[31]	Yi, L., Liang, Z.T., Peng, Y., Yao, X., Chen, H.B. and Zhao, Z.Z. (2012) Tissue-Specific Metabolite Profiling of Alkaloids in Sinomenii Caulis Using Laser Microdissection and Liquid Chromatography-Quadruple/Time of Flight-Mass Spectrometry. Journal of Chromatography A, 1248, 93-103. http://dx.doi.org/10.1016/j.chroma.2012.05.058
[32]	Kupeli, E., Koçar, M., Yesilada, E. and Baser, K.H.C. (2002) A Comparative Study on the Anti-Inflammatory, Antinociceptive and Antipyretic Effects of Isoquinoline Alkaloids from the Roots of Turkish Berberis Species. Life Sciences, 72, 645-657. http://dx.doi.org/10.1016/S0024-3205(02)02200-2
[33]	Taylor-Clarke, T.E., Undem, B.J., Macglashan, D.W., Ghatta, S., Carr, M.J. and McAlexander, M.A. (2007) Prostaglandin-Induced Activation of Nociceptive Neurons via Direct Interaction with Transient Receptor Potential A1 (TRPA1). Molecular Pharmacology, 73, 274-281. http://dx.doi.org/10.1124/mol.107.040832
[34]	Bao, M.M., Cao, Z.F., Yu, D., Fu, S.L., Zhang, G.C., Yang, P., Pan, Y.Y., Yang, B., Han, H.Y. and Zhou, Q.S. (2012) Columbamine Suppresses the Proliferation and Neovascularization of Metastatic Osteosarcoma U2OS Cells with Low Cytotoxicity. Toxicology Letters, 215, 174-180. http://dx.doi.org/10.1016/j.toxlet.2012.10.015
[35]	Chavez, L., Ponce, M.T., Bravo, G. and Pastelin, G. (1989) Pharmacological Study of Dopamine Catabolites on the Contractility of Isolated Guinea Pig Myocardium. Archivos del Instituto de Cardiología de México, 59, 367-373.
[36]	Kuznetsova, N.N., Abdullaeva, L.K. and Sadikov, A.A. (2005) Comparative Action of Salsoline, Salsolidine and Related Compounds on KML Tissue Culture and Animal Tumor Strains. Chemistry of Natural Compounds, 41, 234-235. http://dx.doi.org/10.1007/s10600-005-0121-z
[37]	Yadav, D.K., Singh, N., Dev, K., Sharma, R., Sahai, M., Palit, G. and Maurya, R. (2010) Anti-Ulcer Constituents of Annona squamosal Twigs. Fitoterapia, 82, 666-675. http://dx.doi.org/10.1016/j.fitote.2011.02.005
[38]	Wangchuck, P., Keller, P.A., Pyne, S.G., Wyllis, A.C. and Kamchonwongpaisan, S. (2012) Antimalarial Alkaloids from a Bhutanese Traditional Medicinal Plant Corydalis dubia. Journal of Ethnopharmacology, 143, 310-313.
[39]	Alexander, J., Benford, D., Cockburn, A., Cravedi, J.P., Dogliotti, E., Di Domenico, A., Férnandez-Cruz, M., Fürst, F., Fink-Gremmels, J., Galli, C.L., Grandjean, P., Gzyl, J., Heinemeyer, G., Johansson, N., Mutti, A., Schlatter, J., Van Leeuwen, R., Van Peteghem, C. and Verger, P. (2008) Tropane Alkaloids (from Datura sp.) as Undesirable Substances in Animal Feed. EFSA Journal, 691, 1-55.
[40]	Jacob III, P., Hatsukami, D., Severson, H., Hall, S., Yu, L. and Benowitz, N. (2002) Anabasine and Anatabine as Biomarkers for Tobacco Use during Nicotine Replacement Therapy. Cancer Epidemiology, Biomarkers & Prevention, 11, 1668-1673.
[41]	Hou, Y.Y., Cao, X.L., Wang, L.Q., Cheng, B.F., Dong, L.Y., Luo, X.D., Bai, G. and Gao, W.Y. (2012) Microfractionation Bioactivity-Based Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry for the Identification of Nuclear Factor-κB Inhibitors and β2 Adrenergic Receptor Agonists in an Alkaloidal Extract of the Folk Herb Alstonia scholaris. Journal of Chromatography, 908, 98-104.
[42]	Edwards, M.R., Bartlett, N.W., Clarke, D., Birrell, M., Belvisi, M. and Johnston, S.L. (2009) Targeting the NF-κB Pathway in Asthma and Chronic Obstructive Pulmonary Disease. Pharmacology & Therapeutics, 121, 1-13. http://dx.doi.org/10.1016/j.pharmthera.2008.09.003
[43]	Dias, K.L., Da Silva Dias, C., Barbosa-Filho, J.M., Almeida, R.N., De Azevedo Correia, N. and Medeiros, I.A. (2004) Cardiovascular Effects Induced by Reticuline in Normotensive Rats. Planta Medica, 70, 328-333.
[44]	Medeiros, M.A.A., Nunes, X.P., Barbosa-Filho, J.M., Lemos, V.S., Pinho, J.F., Roman-Campos, D., De Medeiros, I.A., Araújo, D.A.M. and Cruz, J.S. (2008) (S)-Reticuline Induces Vasorelxation through the Blockage of L-Type Ca2+ Channels. Naunyn-Schmiedeberg’s Archives of Pharmacology, 379, 115-125. http://dx.doi.org/10.1007/s00210-008-0352-1
[45]	Cheng, P., Ma, Y., Yao, S., Zhang, Q., Wang, E., Yan, M., Zhang, X., Zhang, F. and Chen, J. (2007) Two New Alkaloids and Active Anti-Hepatitis B Virus Constituents from Hypserpa nitida. Bioorganic & Medicinal Chemistry Letters, 17, 5316-5320. http://dx.doi.org/10.1016/j.bmcl.2007.08.027
[46]	Elgorashi, E., Stafford, G.I. and Van Staden, J. (2004) Acetylcholinesterase Enzyme Inhibitory Effects of Amaryllidaceae Alkaloids. Planta Medica, 70, 260-262. http://dx.doi.org/10.1055/s-2004-818919
[47]	Verotta, L., Orsini, F., Sbacchi, M., Scheildler, M.A., Amador, T.A. and Elisabetsky, E. (2002) Synthesis and Antinociceptive Activity of Chimonanthines and Pyrrolidinoindoline-Type Alkaloids. Bioorganic & Medicinal Chemistry, 10, 2133-2142. http://dx.doi.org/10.1016/S0968-0896(02)00078-0